Membrane fusion at neuronal synapses is a highly regulated process that is triggered by micromolar concentrations of calcium in the presynaptic cell. The SNARE proteins drive fusion and form the critical core of the fusion machinery; however, they are not directly regulated by calcium. Synaptotagmin 1 is a vesicle associated membrane protein that functions as the calcium sensor in neuronal exocytosis. It is anchored by a single transmembrane segment at its N-terminus and contains two C2 domains that have been shown to bind membranes in a calcium-dependent fashion. Synaptotagmin also interacts with SNAREs. At the present time, the nature of the synaptotagmin interactions that mediate fusion are not understood. The proposed work will utilize evolving magnetic resonance methods, such as site-directed spin labeling and pulse EPR, to evaluate a number of proposed mechanisms for the molecular function of sytnaptotagmin. The specific aims are to test the importance of a bridging conformation that is observed for synaptotagmin 1, where the two C2 domains bind opposing bilayers. Proposed experiments will test the hypothesis that synaptotagmin 1 switches from SNARE to membrane interactions in the presence of calcium, and both membrane and SNARE associated structures for synaptotagmin 1 will be generated. A final aim will examine the effects of synaptotagmin 1 on lipid bilayer properties and test the role of these effects in mediating membrane fusion. Membrane fusion is central to cellular processes such as intracellular transport, host defense (killing of microorganisms, immune response), and human physiology and disease (e.g., glucose regulation/diabetes, allergic response). Calcium -triggered neuronal fusion is a central process in neurobiology and it underlies synaptic transmission and potentiation. Regulated neuronal exocytosis is thought to be relevant to an understanding of schizophrenia, bipolar disorder, Huntington's disease, Parkinson's disease, Alzheimer's disease as well as range of other neurological disorders.